The broad, long-term objectives of this application are: 1) to investigate the mtDNA structural alterations occurring with age in human fibroblasts from a large set of individuals 20 weeks (fetal) to 103 years of age, in order to identify mtDNA domains which are hot spots for aging-related mutations, and to determine the frequency of these mutations and the variability among individuals in aging-dependent mtDNA damage; 2) to carry out a detailed functional analysis of the fibroblasts from the individual mitochondrial donors with the aim of establishing the occurrence of any statistically significant correlation between severity of the observed functional defects and the frequency of aging-dependent sets of mutations occurring in different domains of mtDNA; 3) to investigate the pattern of segregation of these mutations during in vitro growth of the fibroblasts and after transfer of mitochondria from the latter into human mtDNA-less (p/0) cell lines; and 4) to investigate the structural and functional alterations occurring in mtDNA of synaptic mitochondria from different regions of human brain. Specifically, the present proposal aims at screening the fibroblast DNA samples by denaturant gradient gel electrophoresis (DGGE) for detection of the cumulative damage in the mtDNA control regions, or in different tRNA genes or in selected portions of various protein coding genes; this preliminary screening will be followed by cloning of the PCR-amplified putative mutated mtDNA fragments, secondary screening by DGGE for identifying the variant clones and, finally, sequencing of mutant clones. Selected fibroblast cultures whose mtDNA exhibits evidence of aging-related accumulation of mutations and selected cultures lacking such mutations will be subjected to a detailed analysis of their mitochondrial protein synthesis, mtDNA transcription, overall respiration capacity, activity of respiratory complexes, respiratory flux control and oxidation phosphorylation, and production of reactive oxygen species. Furthermore, it is planned to investigate by the approaches described above the aging-related structural and functional alterations of mtDNA occurring in synaptic mitochondria from different brain cortical regions, basal ganglia, substantia nigra and cerebellum. To this end, synaptosomes will be isolated from frozen brain samples, and either analyzed directly, or fused with mtDNA-less derivatives of a neuroblastoma or an osteoblastoma cell line for the subsequent structural and functional characterization of the derived transformants. These experiments are expected to provide crucial insights into the role of the accumulation of mtDNA mutations in the aging processes in a proliferating or a postmitotic cell type, with significant implications for the possible involvement of the decay of oxidative phosphorylation in late-onset degenerative diseases.